Project Summary Major Depressive Disorder (MDD) is the most common psychiatric disorder with a lifetime prevalence of 1 in 5, treatment for which is currently suboptimal. Deficits in ability to cope with ongoing chronic stressors are one of the chief predisposing factors to MDD. Neurons of the Paraventricular Thalamus (PVT) are critical in emotional arousal and orchestrate adaptive stress-relevant neurohumoral responses. Furthermore, a synaptic target of the PVT, the oval nucleus of the Bed Nucleus of the Stria Terminals (BNSTov), regulates behavioral responses to stress through Corticotropin-Releasing Factor (CRF)-releasing neurons. The PVT is critical in regulating relevant neurohumoral responses to chronic stress, thought to occur via CRF-neurons of a PVT-BNSTov circuit. Additionally, I have preliminary electrophysiological and single-cell qRT-PCR data in the rat and tissue micropunches in the mouse, that chronic stress accompanies increases in neuronal excitability and enhancement of crf and crf receptor 1 gene expression selectively in the BNSTov. Given that these cells are under direct PVT synaptic influence, this could represent a PVT-mediated adaptive response. Interestingly, unlike in acute stress, in chronic stress, PVT mediated adaptive neural responses appear to be temporally independent of the actual behavioral expression of that stress adaptation. Therefore, this PVT-BNSTov circuit may underlie the latency in the conversion of chronic stress into depressive-like behaviors. To determine the time course wherein stress-induced depressive-like behavior emerges, I employed the Chronic Social Defeat Stress (CSDS) paradigm in mice, and conducted social interaction/sucrose preference testing at various stress duration lengths of 4-10 days. I observed that between 7 and 10 days of CSDS, animals underwent a switch from social approach (?resilient?) to socially avoidant (?susceptible?) phenotypes. The electrophysiological and molecular substrates underlying this rapid behavioral shift remains unknown. I hypothesize that PVT-BNSTov connectivity may govern the temporal emergence of stress susceptibility in the face of persistent psychosocial stress, through PVT synaptic influence on CRF BNSTov neurons. I plan to investigate this functional PVT-BNSTov circuit using electrophysiological, single-cell transcriptomics, and advanced optical circuit dissection methods (DREADDs). First, I will establish functional connectivity by correlating tract tracing and c-Fos activity with the co-occurrence over the time period capturing the behavioral switch (aim 1). Then, I will record in ex-vivo whole-cell patch clamp the intrinsic neuronal properties and procure the cytoplasm for single-cell qRT-PCR analysis in cells before or after the behavioral transition (aim 2). Lastly, I will establish the necessity of the PVT-BNST circuit in mediating the temporal onset of susceptibility via time- and circuit-specific manipulations using DREADDs (aim 3). This proposed state-of-the-art circuit- and cell-type specific manipulations will provide novel insight into stress insult-dependent mechanisms that underlie MDD etiology, which will give rise to more effective therapeutic strategies for MDD.